Epigenome-wide exploratory study of blood DNA methylation and markers of physical performance indicates subtle differences across levels of cardiovascular fitness

Introduction: Exercise is known to exert systemic effects associated with improved cardiovascular fitness and epigenetic modifications. However, the relationships between systemic epigenetic markers and physical fitness indicators remain insufficiently characterized. Methods: In the present study, the Illumina MethylationEPIC array (~730,000 CpG sites) was used to investigate associations between blood DNA methylation and key continuous fitness indicators-estimated VO₂max, grip strength, and vertical jump height-in a cohort of healthy adults. Epigenome-wide association analyses were adjusted for age, sex, estimated blood cell composition, and multiple testing. Results: No significant single-CpG associations were identified after rigorous adjustment. However, when participants were stratified into groups based on age- and sex-specific VO₂max norms, three CpG sites demonstrated differential methylation at a false discovery rate threshold (FDR < 0.05). These included cg21308111 in TNFRSF10A and cg17890932 in USP24, both showing higher methylation in lower-fitness groups, and an intergenic CpG site (cg02967877) exhibiting the opposite pattern. Region-based analyses further identified several differentially methylated regions (DMRs) between fitness groups. Discussion: These findings suggest that variation in cardiorespiratory fitness may be associated with subtle alterations in blood DNA methylation, particularly at loci related to metabolic regulation, inflammatory pathways, and intercellular communication. These findings shouldbe considered hypothesis-generating and require confirmation in larger, independent cohorts and, ideally, in longitudinal or intervention studies to elucidate more directly how changes in physical fitness relate to changes in DNA methylation patterns.